Abstract The tumor suppressor protein p53 is genetically inactivated in ~50% of human tumors. In tumor cells with wild type p53, however, p53 activity is often suppressed in various other ways. In this regard, the ubiquitin E3 ligase Mdm2 and its homolog, MdmX, are critical for the suppression of the activity of p53. Mdm2 and MdmX form heterodimers which catalyze p53 protein degradation and block its transcriptional activity. Consequently, elimination of both Mdm2 and MdmX is essential for full p53 activation. Despite numerous studies on the regulation of p53 activity, the molecular mechanism that mediates the Mdm2-MdmX heterodimer degradation remains poorly understood particularly in vivo. We have recently obtained results strongly indicating that the HECT-domain ubiquitin E3 ligase Huwe1 may be the long-sought E3 ligase responsible for DNA damage- induced degradation of both Mdm2 and MdmX. Importantly, Huwe1 was initially discovered as an E3 ligase that ubiquitinates p53 for degradation. Nevertheless, our preliminary data show that: (a) Huwe1 interacts with both Mdm2 and MdmX proteins independently of p53; (b) DNA damage-induced degradation of Mdm2 and MdmX was markedly inhibited upon Huwe1 siRNA-mediated knockdown; and (c) Huwe1 knockdown renders cells highly resistant to DNA damage-induced p53 activation by stabilizing both Mdm2 and MdmX. We hypothesize that while suppressing steady-state p53 levels under non-stressed conditions, Huwe1 activates p53 by promoting the degradation of Mdm2 and MdmX in response to genotoxic stress, an event critical for p53 activation. To test this hypothesis in vivo, we created conditional Huwe1 knockout mouse models. Using the mouse models, we will determine how acute deletion of Huwe1 impacts p53 activation before and after DNA damage (Aim 1) and identify the impact of Huwe1 loss in DNA damage-induced thymic lymphomagenesis (Aim 2). The successful completion of the proposed in vivo studies will provide the proof-of-concept evidence for the role of Huwe1 as a crucial activator of p53 upon DNA damage. These outcomes will change the prevailing view of this controversial E3 ligase and will also provide a rationale for revising the regulation of Mdm2/MdmX stability and impetus for investigating the mechanism of Huwe1 regulation in the future.